ABSTRACT

Replication and transcription activator (RTA), an immediate-early gene, is a key molecular switch to evoke lytic replication of gammaherpesviruses. Open reading frame 49 (ORF49) is conserved among gammaherpesviruses and shown to cooperate with RTA in regulating virus lytic replication. Here we show a molecular mechanism and in vivo functions of murine gammaherpesvirus 68 (MHV-68 or γHV-68) ORF49. MHV-68 ORF49 was transcribed and translated as a late gene. The ORF49 protein was associated with a virion, interacting with the ORF64 large tegument protein and the ORF25 capsid protein. Moreover, ORF49 directly bound to RTA and its negative cellular regulator, poly(ADP-ribose) polymerase-1 (PARP-1), and disrupted the interactions of RTA and PARP-1. Productive replication of an ORF49-deficient mutant virus (49S) was attenuated in vivo as well as in vitro. Likewise, latent infection was also impaired in the spleen of 49S-infected mice. Taken together, our results suggest that the virion-associated ORF49 protein may promote virus replication both in vitro and in vivo by providing an optimal environment in the early phase of virus infection as a derepressor of RTA.

INTRODUCTION

Gammaherpesviruses replicate in epithelial cells and establish latency mainly in lymphocytes. Two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), were identified to date and both are known as causative agents of various kinds of tumors. EBV is related to Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma (30), while KSHV is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (10). Murine gammaherpesvirus-68 (MHV-68 or γHV-68) is a natural pathogen of small rodents and considered to be an important small animal model system for the study of human gammaherpesviruses due to its high homology in genome sequences and amenable experimental systems both in vitro and in vivo (27).

All herpesviruses share a characteristic virion structure, which is composed of the nucleocapsids, the envelope, and the tegument. The nucleocapsids of icosahedral symmetry contain the viral DNA core and the capsid proteins (29). The outermost portion of a virus particle is the envelope. It contains the lipid layers and many glycoproteins that are important for virus entry (1). The tegument is an electron-dense structure existing between the nucleocapsids and the envelope and constructed with viral- and cellular-encoded proteins and RNAs (26). The tegument proteins are carried into newly infected cells as already-synthesized proteins to immediately activate viral gene promoters and modulate host environments favorable for virus replication by shutting down host protein synthesis and inhibiting infection-triggered immune responses (29). Furthermore, the tegument proteins are involved in transportation of the nucleocapsids into the nucleus (24) as well as in its egress (12, 40). A recent study showed that there is a hub tegument protein interacting with the capsids as well as other tegument proteins (31).

Like other herpesviruses, gammaherpesviruses have two distinct phases of the virus life cycle: productive lytic replication and dormant latent infection. During latent infection, viral genomes are maintained as episomes and only a small subset of viral genes are expressed (29, 43). During lytic replication, viral genes are fully expressed in a tightly regulated manner and infectious virions are produced. Although it is latent infection that allows herpesviruses to establish lifelong persistent infection, lytic replication also contributes to the maintenance of the latent reservoir by transmitting infectious virus particles within the host and among the hosts upon reactivation. Replication and transcription activator (RTA) of gammaherpesviruses plays a pivotal role in initiation of viral lytic replication and reactivation from latency. The expression of RTA is necessary and sufficient for induction of lytic replication (22, 23, 36). To date, various cellular factors have been reported to regulate the RTA activity (as reviewed in references 8 and 35). While some cellular factors such as Sp1, Sp3, octamer-binding protein (Oct-1), CCAAT/enhancer binding protein α (C/EBP-α), Ap-1, K-RBP, and RBP-J-κ are known to positively regulate RTA, poly(ADP-ribose) polymerase-1 (PARP-1), Ste20-like kinase hKFC, histone deacetylase 1 (HDAC1), and interferon regulatory factor 7 (IRF-7) repress RTA transactivation (6, 13–15, 37–39, 42). PARP-1 is a multifunctional protein which involves differentiation, proliferation, tumor transformation, and DNA damage recovery (19, 20). PARP-1 interacts with and downregulates the transcriptional activity of RTA via poly(ADP-ribosyl)ating RTA, which leads to overall repression of viral lytic replication (15). Since PARP-1 expression is relatively abundant in most cells, it is not clear how RTA overcomes repression by PARP-1 at an initial phase of virus infection.

Open reading frame 49 (ORF49) is conserved among gammaherpesviruses and located adjacent to the ORF50 locus in the genome (11, 17, 21). All ORF49 homologs, such as EBV BRRF1 (also called Na) and ORF49 of KSHV and MHV-68, have been shown to cooperate with RTA in regulating virus replication, suggesting an important function of ORF49 as a viral factor that may positively regulate RTA (11, 17, 21). However, the underlying molecular mechanisms of how ORF49 facilitates RTA function remain unclear. Here we characterized ORF49 expression during de novo replication and investigated its virion association. We also studied a molecular mechanism of ORF49 as a derepressor of RTA and the effects of ORF49 deficiency on virus replication of MHV-68 in cultured cells and in mice. Our results highlight the functional importance of ORF49 as a positive viral regulator of RTA during infection in vivo as well as in vitro.

Plasmids.pCMV2-FLAG/ORF49 and pEGFP/ORF49 (where EGFP is enhanced green fluorescent protein) were constructed as described previously (21). All other expression plasmids of MHV-68 ORFs except pCMV2-FLAG/RTA described in this study were prepared in pENTR and constructed into appropriate destination vectors using Gateway technology (Invitrogen) according to the manufacturer's instructions. Particularly, a Myc-tagged ORF49 was generated using a modified version of the pCS3-MT plasmid as a destination vector containing the 6× Myc tag with additional sequences (a gift from Jin-Hyun Ahn, Sungkyunkwan University, Republic of Korea). To introduce mutations into MHV-68, a shuttle plasmid based on pGS284 (kindly provided by Greg Smith at Northwestern University) was constructed. The translational stop codons at the ORF49 locus were introduced by a two-step PCR approach. The sequences upstream (nucleotides [nt] 66961 to 67320) of the stop codons were amplified by primers 49AF (5′-acgcgtcgactccacaatcgcttctaactc-3′) and 49AR (5′-CTGCAGTTAATTAATTGACCGGactgggctgtcaaagtccag-3′) and the downstream (nt 67321 to 67680) by primers 49BF (5′-cccagtCCGGTCAATTAATTAACTGCAGaaattgacagtgcctatggcc-3′) and 49BR (5′-ccttgcatgctgcttcgatccgtgccagtcctataagaac-3′). The uppercase letters indicate the inserted nonviral sequences, including PacI and PstI sites. In a subsequent PCR, two PCR products were mixed as templates and amplified with primers AF and BR. The final PCR products were cloned into pGS284 using SalI and SphI sites (pGS284/49S). The wild-type sequences of ORF49 (nt 66961 to 67680) were amplified with primers AF and BR using bacterial artificial chromosome (BAC) DNA of MHV-68 as a template and cloned into pGS284 to generate a marker rescue (49S-MR).

Generation of 49S and 49S-MR viruses.The recombinant MHV-68 BAC plasmids of 49S and 49S-MR were generated by the two-step allelic exchange method described by Smith and Enquist (33). Insertion or removal of stop codons were screened by PCR and restriction enzyme digestion and confirmed by sequencing. The genome integrity of positive clones was further examined by restriction enzyme digestion and Southern blot analysis, as described previously (41). A BAC plasmid of 49S or 49S-MR was reconstituted in BHK-21 cells by cotransfecting a Cre recombinase expressing plasmid using Lipofectamine Plus (Invitrogen) to excise the BAC sequences. The genome integrity of the produced viruses was determined by restriction enzyme digestion and Southern blot analysis and their titers were measured by plaque assays.

Northern blot analysis.For expression kinetic assays, 5 × 105 of BHK-21 cells were seeded to a 6-well plate at 1 day prior to infection. The cells were infected with the wild-type MHV-68 at a multiplicity of infection (MOI) of 5 and viral inoculums were removed 1 h after infection. Cycloheximide (400 μg/ml; Sigma) was added to cells 1 h before and after virus inoculation for 8 h. Phosphonoacetic acid (200 or 400 μg/ml; Sigma) was added to the cells at 1 h after virus inoculation for 24 h. Total RNAs were extracted by TRI reagent (Molecular Research Center) by following the manufacturer's instructions. For Northern blot analysis, extracted RNAs were analyzed by agarose-formaldehyde gel electrophoresis and transferred to a nylon membrane (Amersham Biosciences). Radiolabeled probes specific to ORF49 or RTA were generated by the random priming method using the ORF49 PCR products (nt 67012 to 67519) or the ORF50 PCR products (nt 68647 to 69178), respectively, as a template with [α-32P]dCTP. A radiolabeled β-actin probe was used as a control blot. The signals were detected and analyzed using a multiplex bioimaging system (FLA-7000; Fujifilm).

Antibodies and Western blot analysis.To generate polyclonal antibodies against viral proteins, glutathione S-transferase (GST)-tagged ORF45, ORF49, or ORF65 (M9) was expressed at 18°C in BL21 (DE) cells after overnight induction with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The soluble GST-tagged protein was purified by a GSTrap column (GE Healthcare) and concentrated by Amicon Ultra-15 (Millipore) according to the manufacturer's instructions. Antisera of immunized rabbits were obtained (AbFrontier) and tested for their specificity. For Western blot analysis, whole-cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose or polyvinylidene fluoride membrane. Proteins were probed with primary antibodies against ORF45 (1:1,000), ORF49 (1:500), M7 (1:500), ORF65 (M9) (1:1,000), FLAG (1:2,000; Sigma), and PARP-1 (1:500; BD Pharmingen). Anti-M7 (gp150) polyclonal antibody was kindly provided by James Stewart (University of Liverpool, United Kingdom). Goat anti-rabbit or goat anti-mouse IgG conjugated with horseradish peroxide secondary antibody (Santa Cruz) was detected by an EPD Western blot detection kit (ELPIS, Republic of Korea), and the signals were detected and analyzed using LAS-4000, a chemiluminescent image analyzer (Fujifilm). Protein sizes based on amino acid sequences were predicted by Science Gateway.

Coimmunoprecipitation.Approximately 4 × 106 293T cells were seeded into 100-mm plates, transfected with plasmid DNAs by polyethylenimine (4), and incubated for 48 h. Cells were scraped and resuspended in the IP buffer (20 mM HEPES [pH 7.4], 100 mM NaCl, 0.5% Nonidet P-40, and 1% Triton X-100) supplemented with 1/100 volume of protease inhibitor cocktail (Sigma) and 1 mM PMSF. Cells were rotated at 4°C during 1 h, and cell debris was removed by centrifugation (12,000 × g, 4°C, 10 min). The cell lysates were incubated at 4°C with an appropriate antibody for 1 h and further incubated with protein A/G agarose beads (Pierce) overnight at 4°C. The beads were washed 3 to 5 times by IP buffer and subjected to Western blot analysis.

Multiple- or single-step growth curves of viruses.The replication kinetics of the wild type (WT), 49S, and 49S-MR were assayed in MEF cells. The cells were incubated with viral inoculums for 1 h at an MOI of 0.05 or 2 for multiple- or single-step growth curves, respectively. After 1 h of incubation, the inoculums were removed and the cells were washed three times with PBS and added with fresh medium. The cells and the supernatants were harvested together at various times points and subjected to three cycles of freezing and thawing. The lysates were cleared by low-speed centrifugation to remove cell debris, and then the virus titers of the supernatants were analyzed by plaque assays.

Mice experiments.All animal experiments were approved by the Korea University Institutional Animal Care & Use Committee (KUIACUC-2009-105) in accordance with institutional guidelines. Six- to eight-week-old BALB/c mice (Samtako, Republic of Korea) were intranasally (i.n.) infected with the wild-type, 49S, and 49S-MR viruses (1,000 PFU/mouse) under the anesthetized condition. Mice were sacrificed at 6 days postinfection for acute infection. The viral loads in the lung and in the spleen were measured as described previously (21, 34). Briefly, for acute infection analysis, the lung tissues were homogenized in 1 ml of DMEM, and the virus titers were determined by plaque assays. For latent viral loads, ex vivo limiting dilution assays were performed. BHK-21 cells (2 × 103/well) were seeded in 96-well plates, and serial 2-fold dilutions of splenocytes, starting with 106 cells/well, were plated onto BHK-21 cells with 24 wells per dilution. After 7 days, each well was scored for cytopathic effects, and the percentage of cytopathic effects per 24 wells was determined per dilution. As a control, the presence of preformed viruses in the splenocytes was also examined by incubating BHK-21 cells with the splenocytes following three rounds of freezing and thawing.

Quantitative real-time PCR.Splenocytes (107) from infected mice were lysed overnight in 20 mM Tris-HCl (pH 7.5), 10 mM EDTA (pH 8.0), 100 mM NaCl, and 0.5% SDS with 500 μg/ml of proteinase K. Viral genomic DNAs were isolated and prepared by phenol-chloroform-isoamyl alcohol (25:24:1) extraction and ethanol precipitation. SYBR green I (Invitrogen) and ORF56 locus-specific primers (21, 34) were used to quantitate the copy number of viral genomic DNAs on the iCycler iQ multicolor real-time PCR detection system (Bio-Rad), and the results were analyzed on Optical system software (Bio-Rad). The standard DNAs were prepared in triplicates with 10-fold serial dilutions of MHV-68 BAC DNA ranging from 2 to 2 × 107 copies and used to generate a standard curve for each real-time PCR.

RESULTS

ORF49 is expressed as a late gene.To characterize ORF49 expression during virus replication, BHK-21 cells were infected with MHV-68 at an MOI of 5 in the presence or absence of cycloheximide (CHX; a protein synthesis inhibitor) (400 μg/ml) or phosphonoacetic acid (PAA; a viral DNA replication inhibitor) (400 μg/ml). The cells were harvested at the indicated time points and total RNAs were subjected to Northern blot analysis. Cycloheximide was used to determine herpesvirus immediate-early genes since they are defined as those expressed without any protein biosynthesis. Herpesvirus early genes are classified by the dependence of their expression on viral protein synthesis (CHX sensitive), while the late genes are classified by the dependence of their expression on viral DNA replication (PAA sensitive). The ORF49 transcripts were initially detected as early as 4 h and peaked at 18 to 24 h postinfection (Fig. 1A). ORF49 expression was sensitive to both CHX and PAA treatments, while transcription of RTA, an immediate-early gene, was not inhibited by CHX and PAA treatments, albeit affected by the PAA treatment. The limited cytotoxicity of CHX and PAA treatments was shown by the levels of β-actin transcripts. To further confirm these results at the protein level, a polyclonal anti-ORF49 antibody was produced by immunizing rabbits with GST-ORF49 fusion protein. The specificity of the antibody was confirmed in 293T cells transfected with a Myc-tagged, a FLAG-tagged, or an EGFP-fused ORF49 expression plasmid and in NIH 3T3 cells infected with the wild-type MHV-68 at an MOI of 0.5 for 72 h (Fig. 1B). Expression of the ORF49 protein was detected from 8 h and abolished by the PAA treatment (200 μg/ml) at 20 h postinfection when NIH 3T3 cells were infected with the virus at an MOI of 5 (Fig. 1C, upper panel). A well-known late gene product, ORF65 (M9, a small capsid protein) showed PAA-sensitive expression kinetics similar to those of ORF49, whereas ORF37 (alkaline exonuclease, an early gene) showed PAA-resistant expression kinetics (Fig. 1C, lower panels). All these results indicate that ORF49 is transcribed and translated as a late gene during lytic replication of MHV-68.

The orf49 gene is expressed as a late gene. (A) Northern blot analysis of orf49 gene expression during de novo infection. BHK-21 cells were infected with MHV-68 at an MOI of 5 and harvested at the indicated time points. Cycloheximide (CHX) and phosphonoacetic acid (PAA) were treated for 8 h and 24 h, respectively. Total RNAs were extracted and analyzed by Northern blot analysis using an ORF49- or RTA-specific radiolabeled probe. The 18S rRNA as well as β-actin transcript was used as a loading control of the samples. RTA is an immediate early gene. (B) Detection of the ORF49 protein by an anti-ORF49 polyclonal antibody. The whole-cell lysates of 293T cells transfected with Myc- or FLAG-tagged ORF49 or EGFP-ORF49 were harvested at 48 h posttransfection and subjected to Western blotting using the anti-ORF49 antibody. The ORF49 protein of NIH 3T3 cells infected with MHV-68 at an MOI of 0.5 for 72 h were detected with the anti-ORF49 antibody. Vector-transfected or mock-infected cells were used as a control. The antibody was generated by immunizing purified GST-ORF49 in rabbits. Arrowheads indicate the specific bands for the ORF49 proteins. (C) Expression kinetics of the ORF49 protein during de novo infection of MHV-68. NIH 3T3 cells were infected with MHV-68 at an MOI of 5. PAA (200 or 400 μg/ml) was treated for 20 h. The cells were harvested at the indicated time points and analyzed by Western blots using antibodies against ORF49, ORF37 (alkaline exonuclease, an early gene), and ORF65 (small capsid protein, a late gene). Tubulin was used as a loading control. An open arrowhead indicates an ORF37-specific band, while the asterisk indicates a nonspecific band, serving as a loading control. IB, immunoblot assay.

ORF49 is associated with the virion.Since MHV-68 ORF49 was shown to cooperate with RTA in regulating virus replication (21), yet expressed as a late gene (Fig. 1), we examined whether ORF49 protein is associated with virions, thereby acting at an early phase of infection. MHV-68 virions were purified by a discontinuous sucrose gradient method and treated with Triton X-100 and/or proteinase K. Similar to known virion proteins such as gp150 (M7, an envelope glycoprotein), ORF45 (a tegument protein), and ORF65 (M9, a small capsid protein), ORF49 was associated with virions (Fig. 2A). Treatment with either Triton X-100 or proteinase K alone did not disrupt the virion association of the ORF49 protein. To further define the detergent sensitivity of ORF49 within the virion, we compared detergent-resistant pellet and detergent-sensitive supernatant fractions after the treatment of 2% Triton X-100 (Fig. 2B). The gp150 envelope protein was found in supernatant fractions. While the ORF45 tegument protein was detected in supernatant and pellet fractions, the ORF49 protein and the ORF65 capsid protein were found only in pellet fractions. When exposed to various detergent conditions, both the ORF49 and the ORF65 proteins were able to maintain virion association even under stringent conditions, such as 2% NP-40 with high salt (HiNa) or 2% Triton X-100 with 4% NP-40 (TxNP), while ORF45 was dissociated under mild conditions of 1% NP-40 plus 0.25% sodium deoxycholate (RIPA) (Fig. 2C). The results suggest that ORF49 is more tightly associated with virions in a capsid-like manner than ORF45, an outer tegument protein.

ORF49 is a virion-associated protein. (A) Virion association of ORF49. Purified virions were treated with 2% Triton X-100 and/or proteinase K. The pellets were analyzed by Western blots using anti-gp150 (glycoprotein), ORF45 (tegument), ORF65 (small capsid protein), or anti-ORF49. (B) Association of ORF49 with the capsids. After the treatment with 2% Triton X-100, virions were centrifuged and analyzed as pellet (P) and supernatant (S) fractions. Purified virions (V) with no treatment were used as a control. (C) Detergent sensitivity of virion-associated ORF49. Purified virions were exposed to various detergent concentrations, such as 2% NP-40 with HiNa, 2% Triton X-100 with 4% NP-40 (TxNP), or 1% NP-40 with 0.25% sodium deoxycholate (RIPA). The pellets were subjected to Western blot analysis.

ORF49 interacts with a large tegument and a capsid protein.As the capsid proteins of MHV-68 have been identified (3) and are highly homologous to other herpesviral capsid proteins (7), we reasoned that ORF49 may be classified as an inner tegument protein which interacts with the capsids and other tegument proteins. To determine protein interactions of ORF49 within the virion, we initially tested possible interactions of ORF49 with 12 virion proteins (ORF8, ORF24, ORF25, ORF26, ORF45, ORF50, ORF52, M7, ORF64, ORF65, ORF73, and ORF75c) by yeast two-hybrid assays (data not shown) and then confirmed the interactions with 4 possible candidates (ORF25, ORF26, ORF64, and ORF65) by coimmunoprecipitation (Fig. 3). The ORF25 major capsid protein strongly bound to ORF49 (Fig. 3A), while the ORF26 triplex protein or the ORF65 small capsid protein showed little or no interactions (Fig. 3B and C). The N terminus (amino acids [aa] 1 to 808) of the ORF25 protein had stronger interactions with ORF49 than its C terminus (aa 777 to 1365). In addition, ORF64, a large tegument protein, interacted with ORF49 via its central (aa 887 to 1521) and C-terminal (aa 1513 to 2457) regions (Fig. 3D). The ORF64 protein of KSHV has been reported to interact with several tegument proteins, playing an important role in virion assembly as a hub for tegument and capsid proteins (31). These results suggest that ORF49 may be packaged into virions as an inner tegument protein through interactions with ORF64 and ORF25.

The ORF49 protein interacts with virion proteins. An ORF49 expression plasmid was cotransfected into 293T cells with a plasmid expressing virion proteins such as ORF25, ORF26, ORF65, or ORF64. At 48 h posttransfection, the cells were lysed and coimmunoprecipitated using the anti-FLAG or anti-GFP antibody as indicated. The samples were analyzed by Western blots with anti-FLAG, anti-V5 or anti-GFP antibodies. The whole-cell lysates were loaded as controls. (A) Interactions of ORF49 and ORF25, a major capsid protein. GFP-ORF49 was cotransfected with FLAG-ORF25a (aa 1 to 808) or FLAG-ORF25b (aa 777 to 1365) into 293T cells. (B) No interactions of ORF49 and ORF65, a small capsid protein. FLAG-ORF49 was cotransfected with V5-ORF65 into 293T cells. (C) No interactions of ORF49 and ORF26, a capsid protein. GFP-ORF49 was cotransfected with FLAG-tagged ORF26. (D) Interactions of ORF49 and ORF64, a tegument protein. GFP-ORF49 was cotransfected with FLAG-tagged ORF64a (aa 1 to 320), ORF64b (aa 315 to 917), ORF64c (aa 887 to 1521), or ORF64d (aa 1513 to 2457) in 293T cells. IP, immunoprecipitation assay.

ORF49 enhances the RTA promoter activity by inhibiting the interactions of RTA and PARP-1.According to the yeast two-hybrid screening results of EBV viral proteins with a human cDNA library, EBV BRRF1 was shown to interact with poly(ADP-ribose) polymerase-4 (PARP-4 or vault-PARP) (5). PARP-4, a homolog of PARP-1, is a protein component of cytoplasmic vault ribonucleoprotein particles with unknown functions (18). Interestingly, the N-terminal region of PARP-4 and the catalytic NAD+-binding domain of PARP-1 share an identity of 28% and a homology of 45%, and the catalytic domain of PARP-1 binds to RTA and negatively regulates RTA function through poly(ADP-ribosyl)ation (15). Moreover, PARP-1 was one of the preliminary hits of ORF49-interacting partners in a yeast two-hybrid screening (S. Lee and R. Sun, unpublished data). Thus, we raised a hypothesis that ORF49 may interact with PARP-1 and interfere with the interactions between PARP-1 and RTA. To examine the interactions of ORF49 with PARP-1, FLAG-ORF49 was transfected into 293T cells, and coimmunoprecipitation was performed using anti-FLAG antibody. The results showed that ORF49 directly interacted with endogenous PARP-1 in 293T cells (Fig. 4A). While FLAG-RTA also bound to endogenous PARP-1 as previously reported, cotransfection of GFP-ORF49 abolished the interactions between RTA and PARP-1 (Fig. 4B). Importantly, GFP-ORF49 directly bound to FLAG-RTA (Fig. 4B), suggesting that interactions of ORF49 with PARP-1 and RTA interfere with interactions of PARP-1 and RTA. Since PARP-1 is a negative regulator of RTA, reduced interactions of PARP-1 and RTA by ORF49 may derepress the RTA activity. In agreement with these results, our previous results showed that ORF49 enhanced RTA-mediated transactivation on ORF57 and M3 promoters (21). Taken together, these results suggest that ORF49 may promote expression as well as function of RTA by interfering with interactions of RTA and PARP-1.

ORF49 interacts with PARP-1 and RTA. (A) Direct binding of ORF49 to PARP-1. FLAG-ORF49 was transfected into 293T cells. At 48 h posttransfection, the cells were lysed and immunoprecipitated with the anti-FLAG antibody. The samples were analyzed in Western blots using anti-PARP-1 and anti-FLAG antibodies. (B) Inhibition of interactions between RTA and PARP-1 by ORF49. FLAG-RTA and/or GFP-ORF49 were cotransfected into 293T cells. The cell lysates were coimmunoprecipitated with anti-FLAG and analyzed in Western blots using anti-PARP-1, anti-GFP, and anti-FLAG antibodies.

ORF49-deficient recombinant virus was attenuated in viral growth in vitro and in vivo.We previously reported that transposon insertion at the ORF49 locus causes attenuation of viral growth in vitro (21, 34). However, we cannot completely exclude the possibility that the 1.2-kb-sized transposon may have unexpected influences on neighboring gene expression. It was also observed that ORF49null virus could lose the transposon following repeated passages in permissive cells (data not shown). To precisely delineate the effect of ORF49 deficiency in virus replication, we constructed an ORF49-deficient recombinant virus (49S) by a conventional two-step allelic exchange method. This mutant virus contains triple stop codons, including a PacI site and an additional PstI restriction site within the ORF49 locus (nt 67321), which leads the premature termination of translation with ORF49 (Fig. 5A). A corresponding marker rescue virus (49S-MR) was also constructed as a control. Characterization of these mutants using restriction enzyme digestion followed by Southern blot analysis is shown in Fig. 5B. Due to the frequent loss of transposons found in ORF49null, the viral genome stability of 49S was checked in every passage of recombinant virus propagation by PCR analysis followed by PstI enzyme digestion. As shown in Fig. 5C, there was no spontaneous revertant of 49S found after 5 passages.

Generation of the ORF49-deficient recombinant virus (49S) and its marker rescue virus (49S-MR). (A) Schematic diagram of the ORF49 locus of the MHV-68 genome. Open reading frames are shown as boxes. Arrowheads of the boxes indicate direction of the transcription. Numbers indicate position of each part within the viral genome. (B) Construction of the 49S and 49S marker rescue (49S-MR) viruses. Triple stop codons were introduced into the ORF49 locus (n = 67,320) using two-step allelic exchange in the MHV-68 BAC clone. BAC DNAs prepared from the wild-type, 49S, and 49S-MR viruses were digested by PacI and EcoRI and resolved in a 0.7% agarose gel. Once reconstituted by transfecting BAC DNAs with the cre expressing plasmid to remove the BAC plasmid sequences, the viral genomes of WT, 49S, and 49S-MR viruses were digested with EcoRI or PacI, resolved in an agarose gel, and subjected to Southern blot analysis using a 32P-labeled ORF49-specific probe. (C) Validation of the viral genome stability. The ORF49 locus of amplified 49S and 49S-MR viruses was amplified, passaged five times, digested with PstI, and resolved in 1% agarose gel. BAC DNAs containing 49S or 49S-MR sequence were used as controls.

Consistent with our previous report on ORF49null virus, the virus growth of 49S was reduced compared with that of WT or 49S-MR in a multiple-step growth curve (MOI of 0.05) in MEF cells (Fig. 6A). Other permissive cell lines, such as BHK-21 and NIH 3T3 cells, were also tested for the viral growth of 49S, and similar results were obtained (data not shown). Infection with a high dose of 49S did not overcome this growth defect, as shown in a single-step growth curve (MOI of 2) (Fig. 6B). The higher MOIs could not be used since the maximum titer of the 49S virus was too low. Reduced plaque sizes of the 49S virus further supported the attenuated growth phenotype of 49S (Fig. 6C). To determine the effect of ORF49 deficiency in acute and latent infections in vivo, we intranasally infected WT, 49S, or 49S-MR (1,000 PFU) into BALB/c mice. Compared with that of WT or 49S-MR, lytic replication of 49S was attenuated in the lungs by ∼50-fold during acute infection (Fig. 7A). Viral latency was also severely affected in the splenocytes of mice infected with 49S compared with that in the splenocytes of mice infected with WT or 49S-MR, as measured by ex vivo reactivation limiting dilution and infectious center assays (Fig. 7B and C). The viral genome loads were also slightly lower in the splenocytes of the mice infected with 49S than in with WT or 49S-MR, although the differences were not statistically significant (Fig. 7D). Three independent experiments were performed and showed similar results. Taken together, our results demonstrate that MHV-68 ORF49 may play a critical role in viral infection in vivo in addition to in vitro lytic replication.

In vitro replication of the ORF49-deficient virus. (A) Multiple-step growth curves of WT, 49S, and 49S-MR. MEF cells were infected with WT, 49S, and 49S-MR at an MOI of 0.05 and harvested at the indicated time points. The virus titers in the cells and the supernatants were analyzed by plaque assays. (B) Single-step growth curves of WT, 49S, and 49S-MR. MEF cells were infected with WT, 49S, and 49S-MR at an MOI of 2 and harvested at the indicated time points. The virus titers in the cells and the supernatants were analyzed by plaque assays. (C) Plaque sizes of WT, 49S, and 49S-MR. Plaque assays were performed on Vero cells with similar titers of each virus, and the diameters of plaques were determined from at least 100 plaques per virus. The average plaque sizes are shown with standard errors. Representative pictures of actual plaques are shown in the lower panel.

In vivo replication of the ORF49-deficient virus. (A) Acute infections of WT, 49S, and 49S-MR in the lung. BALB/c mice (n = 6) were intranasally infected with 1,000 PFU of each virus. At day 6 after infection, the lungs were homogenized to determine lytic viral titers using plaque assays. Each symbol represents the viral titer from the lung tissues of individual mice, with the bar as the mean value. (B to D) Latent infections of WT, 49S, and 49S-MR in the spleen. BALB/c mice (n = 5) were intranasally infected with 1,000 PFU of each virus. At day 14 postinfection, single splenocyte suspensions were prepared and analyzed for the frequency of cells reactivating virus by ex vivo reactivation limiting dilution assays (B) and infectious center assays (C). As for the limiting dilution assays, each symbol represents the average percentage of wells positive for CPE, with standard error of the mean. For infectious center assays, each symbol represents the viral titer from individual mice, with the bar as the mean value. (D) The genomic DNAs extracted from the splenocytes of individual mice were analyzed for the viral genome copy number by real-time PCR. The average viral genome copy numbers are shown with standard errors of the means. P values from the Student t test are indicated in the corresponding figures.

DISCUSSION

The ORF49 homologs of gammaherpesviruses, such as EBV BRRF1, KSHV, and MHV-68 ORF49, have been reported to functionally cooperate with RTA, a key switch molecule of lytic replication (11, 17, 21). However, the molecular mechanisms and in vivo effects of ORF49 remain elusive. We report here that ORF49 cooperates with RTA during an early phase of virus infection as an inner tegument protein by interfering with the interactions of RTA and PARP-1, a negative regulator of RTA. Furthermore, infection of the ORF49-deficient virus resulted in attenuation of acute and latent infection in mice, indicating the functional importance of ORF49 for viral fitness in vivo.

Late gene kinetics and virion association of ORF49.Previous studies on MHV-68 transcriptome using a microarray reported that the MHV-68 ORF49 transcript showed early-late or early kinetics during de novo infection (9, 25). Our Northern blot results showed that expression of the ORF49 transcript started from 4 h and peaked at 18 to 24 h postinfection. While the RTA transcript was cycloheximide resistant, the ORF49 transcript was cycloheximide and phosphonoacetic acid sensitive (Fig. 3A). We also showed that the PAA treatment abolished the ORF49 protein expression, similar to the case of the ORF65 small capsid protein (Fig. 3C). Given its expression kinetics and drug sensitivity in Northern blot and Western blot analyses, ORF49 can be classified as a late gene during de novo infection. These results are seemingly paradoxical when considering the function of ORF49 in cooperating with RTA, an immediate-early gene. Now that the ORF49 protein is found to be a virion component, it is conceivable that ORF49, a late gene product, may act as a tegument protein to promote RTA function at an early phase of virus infection. Interactions of ORF49 with the ORF64 hub tegument protein and the ORF25 capsid protein may facilitate the association of ORF49 with the virion as an inner tegument. In addition, interactions of ORF49 with the nucleocapsids may facilitate the delivery of the ORF49 protein near the nucleus, so that it can be efficiently transported through the nuclear pore complex into the site of replication. Consistent with our finding, ORF49 of rhesus monkey rhadinovirus was reported to be a virion protein by mass spectrometric analyses (28), suggesting a conserved important function of ORF49 as a virion component during gammaherpesvirus replication.

Molecular functions and mechanisms of the ORF49 protein.The ORF49 homologs of EBV, KSHV and MHV-68 have been shown to enhance lytic replication in cooperation with RTA (11, 17, 21). While molecular mechanisms remain unclear, ORF49 molecular functions seem to differ in gamma-1 (EBV) and gamma-2 (KSHV and MHV-68) herpesviruses. The EBV BRRF1 (Na protein) alone was able to stimulate the immediate-early gene BZFL1 (ZEBRA or Z) promoter containing a c-Jun binding site in EBV-negative cells (11, 17, 21). Although KSHV and MHV-68 ORF49 could facilitate RTA-mediated transactivation in a number of lytic promoters, these ORF49 proteins lacked the ability to transactivate by themselves (11, 17, 21). The EBV Na protein was predominantly found in the nucleus (32), whereas KSHV and MHV-68 ORF49 were evenly distributed both in the nucleus and the cytoplasm. Based on time course of expression, EBV Na and KSHV ORF49 were shown to be early genes (11, 17, 21), but MHV-68 ORF49 was classified as a late gene based on the drug sensitivity and the expression kinetics at the transcript and the protein levels (Fig. 1). These discrepancies may reflect the distinct mechanisms of gamma-1 and gamma-2 herpesviruses in regulation of viral lytic replication. While we were preparing our manuscript, a recent study was reported that overexpression of the Na protein was sufficient to induce lytic replication in EBV-positive epithelial cells in a TRAF2- and p53-dependent manner (16).

Here we provide evidence for a molecular mechanism that MHV-68 ORF49 may function as a derepressor of RTA to facilitate RTA transactivation. PARP-4 of currently unknown function is a homologous protein of PARP-1, and PARP-4 was reported to interact with EBV BRRF1 (5). PARP-1 was also identified as an interacting partner of ORF49 in our yeast two-hybrid screening. Since PARP-1 was shown to be a repressor of RTA, we investigated the interactions of MHV-68 ORF49 and PARP-1 in coimmunoprecipitation assays. The direct interactions of ORF49 and PARP-1 ablated the interactions of RTA and PARP-1. These results suggest that ORF49 may function as a depressor of RTA, thereby optimizing virus lytic replication during early infection. Interestingly, ORF49 also interacted with RTA, suggesting another plausible mechanism by which ORF49 may directly promote RTA function in a PARP-1-independent manner. These results are consistent with our previous finding that overexpression of ORF49 increased viral lytic gene expressions and virion production (11, 17, 21). A schematic diagram of a virion-associated ORF49 as a positive viral regulator of RTA is depicted as our working hypothesis in Fig. 8.

A schematic diagram of ORF49 as a derepressor of RTA during de novo infection. In the absence of ORF49, PARP-1 may interact with RTA and negatively regulates RTA function through poly(ADP-ribosyl)ation of RTA, thereby inhibiting the transactivation of viral lytic promoters. Upon virus infection, the inner tegument protein ORF49 is transferred into the nucleus where ORF49 promotes RTA-mediated transactivation of the lytic promoters by inhibiting the interactions of RTA with PARP-1, shown with a (−) symbol. In addition, the ORF49 protein itself may directly bind to RTA to facilitate RTA function regardless of PAPR-1 inhibition, shown with a (+) symbol. As indicated with a thicker arrow in the forward direction, the balance between repression (left side) or derepression (right side) of RTA by PARP-1 may favor the forward direction in the presence of the ORF49 protein during de novo infection.

Critical functions of ORF49 during MHV-68 infection in mice.MHV-68 provides an efficient genetic system to study virus infection and pathogenesis of human gammaherpesviruses in vivo. Thus, we sought to examine in vivo functions of ORF49 using MHV-68 infection using a newly generated ORF49-deficient virus (49S) containing premature translation stop codons. In agreement with our previous results of the transposon-inserted ORF49null virus, the viral growth of the recombinant 49S virus was attenuated in cell culture with smaller-sized plaques (Fig. 6). This attenuation in virus replication in vitro may account for attenuation in acute lytic replication of the 49S virus in the lung. As measured by ex vivo reactivation assays, latent infection was also attenuated in the splenocytes of 49S-infected mice. The lower level of latently infected cells found in 49S-infected mice may be likely due to attenuated acute replication in the lung, which also argues for the important contribution of lytic replication to latent infection. Alternatively, it may be owing to the reduced ability of 49S to establish latency in splenocytes or to efficiently reactivate from latently infected splenocytes. Taken together, our results highlight the functional importance of ORF49 in MHV-68 infection in vivo.

In conclusion, we found that MHV-68 ORF49 contributes to the virus life cycle at least in two ways. First, ORF49 promotes viral growth by derepressing the PARylated RTA, thereby further enhancing RTA function and expression. Second, ORF49 is packaged into a virion as an inner tegument protein, which allows the ORF49 protein to act on RTA during the early phase of de novo infection. Although more studies should be warranted to understand molecular mechanisms by which ORF49 may affect viral replication during reactivation, our work may offer a clue to understand functional mechanisms of ORF49 in regulating viral replication in vitro and in vivo.

ACKNOWLEDGMENTS

This work was supported by grants from the Korea Research Foundation funded by the Korean government (MOEHRD) (KRF-2007-531-C00044), the National R&D Program for Cancer Control, Ministry of Health, Welfare and Family Affairs, Republic of Korea (0920170), and the Mid-career Researcher Program through the NRF funded by the MEST (NRF-2010-0000484).

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